Benson M. George

PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin’s lymphoma. Although it is well established that PD-1–PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1–PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1–PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors

Significance This is the first in-depth profiling of pancreatic neuroendocrine tumors (PanNETs), to our knowledge, that illuminates fundamental biological processes for this class of tumors. Beginning with the index case and confirmed with additional patient tumors, we showed the dependence on paracrine signaling through the hepatocyte growth factor (HGF)/receptor tyrosine kinase MET axis. We created a novel cell line derived from a well-differentiated PanNET with autocrine HGF/MET signaling. We also identified the cell-surface protein CD90 as a marker of the tumor-initiating cell population in PanNETs that allows for prospective isolation of this critical cell population. Finally, we demonstrated the efficacy of anti-CD47 therapy in PanNETs. These findings provide a foundation for developing therapeutic strategies that eliminate tumor-initiating cells in PanNETs and show how deep examination of individual cases can lead to potential therapies. Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a “don’t eat me” signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90hi cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes

Engineering cytokine-receptor pairs Interleukin-2 (IL-2) is an important cytokine that helps T cells destroy tumors and virus-infected cells. IL-2 has great therapeutic promise but is limited by toxic side effects and its capacity to both activate and repress immune responses. Sockolosky et al. set out to improve IL-2–based immunotherapy by engineering synthetic IL-2–receptor pairs (i.e., IL-2 and its receptor, IL-2R) (see the Perspective by Mackall). Engineered complexes transmitted IL-2 signals but only interacted with each other and not with endogenous IL-2/IL-2R. Treatment of mice with IL-2 improved the ability of engineered T cells to reject tumors with no obvious side effects. This type of approach may provide a way to mitigate toxicities associated with some cytokine-based immunotherapies. Science, this issue p. 1037; see also p. 990 Engineered cytokines are able to improve immunotherapy in mouse tumor models. Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

Engagement of MHC class I by the inhibitory receptor LILRB1 suppresses macrophages and is a target of cancer immunotherapy

Exciting progress in the field of cancer immunotherapy has renewed the urgency of the need for basic studies of immunoregulation in both adaptive cell lineages and innate cell lineages. Here we found a central role for major histocompatibility complex (MHC) class I in controlling the phagocytic function of macrophages. Our results demonstrated that expression of the common MHC class I component β2-microglobulin (β2M) by cancer cells directly protected them from phagocytosis. We further showed that this protection was mediated by the inhibitory receptor LILRB1, whose expression was upregulated on the surface of macrophages, including tumor-associated macrophages. Disruption of either MHC class I or LILRB1 potentiated phagocytosis of tumor cells both in vitro and in vivo, which defines the MHC class I–LILRB1 signaling axis as an important regulator of the effector function of innate immune cells, a potential biomarker for therapeutic response to agents directed against the signal-regulatory protein CD47 and a potential target of anti-cancer immunotherapy.Host cells display ‘don’t eat me’ signals to protect themselves from phagocytosis. Maute and colleagues identify a novel ‘don’t eat me’ system based on recognition of MHC class I by the phagocyte-expressed inhibitory molecule LILRB1.

Anti-SIRPα antibody immunotherapy enhances neutrophil and macrophage antitumor activity

Significance Cancer immunotherapy is a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. Although cells of the myeloid lineage frequently infiltrate tumors and limit therapeutic success, currently approved immunotherapies primarily target tumor-infiltrating T and natural killer lymphocytes. The inhibitory receptor signal regulatory protein-α (SIRPα) represents a myeloid-specific immune checkpoint that engages the “don’t eat me” signal CD47. Here, we developed an anti-human SIRPα antibody, KWAR23, which in combination with tumor-opsonizing antibodies, greatly augmented neutrophil and macrophage antitumor activity in vitro and in vivo. Thus, KWAR23 may represent a promising candidate for combination therapies and may achieve durable responses in a greater number of patients with cancer. Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-α (SIRPα) is a myeloid-specific immune checkpoint that engages the “don’t eat me” signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human SIRPA knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt’s lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRPα antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRPα blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.

Normal and Neoplastic Stem Cells

A stem cell is broadly defined as a cell that retains the capacity to self-renew, a feature that confers the ability to continuously make identical daughter cells or additional cells that will differentiate into downstream progeny. This highly regulated genetic program to retain “stemness” is under active investigation. Research in our laboratory has explored similarities and differences in embryonic, tissue-specific, and neoplastic stem cells and their terminally differentiated counterparts. In this review, we will focus on the contributions of our laboratory, in particular on the studies that identified the mouse hematopoietic stem cell (HSC) and the human leukemic stem cell. These studies have led to significant improvements in both preclinical and clinical research, including improved clinical bone marrow transplantation protocols, isolation of nonleukemic HSCs, a cancer immunotherapy currently in clinical trials, and development of a HSC reporter mouse. These studies and the current follow-up research by us and others will continue to identify the properties, function, and regulation of both normal and neoplastic stem cells.

Effects of mechanical loading on cortical defect repair using a novel mechanobiological model of bone healing

Mechanical loading is an important aspect of post-surgical fracture care. The timing of load application relative to the injury event may differentially regulate repair depending on the stage of healing. Here, we used a novel mechanobiological model of cortical defect repair that offers several advantages including its technical simplicity and spatially confined repair program, making effects of both physical and biological interventions more easily assessed. Using this model, we showed that daily loading (5N peak load, 2Hz, 60 cycles, 4 consecutive days) during hematoma consolidation and inflammation disrupted the injury site and activated cartilage formation on the periosteal surface adjacent to the defect. We also showed that daily loading during the matrix deposition phase enhanced both bone and cartilage formation at the defect site, while loading during the remodeling phase resulted in an enlarged woven bone regenerate. All loading regimens resulted in abundant cellular proliferation throughout the regenerate and fibrous tissue formation directly above the defect demonstrating that all phases of cortical defect healing are sensitive to physical stimulation. Stress was concentrated at the edges of the defect during exogenous loading, and finite element (FE)-modeled longitudinal strain (εzz) values along the anterior and posterior borders of the defect (~2200με) was an order of magnitude larger than strain values on the proximal and distal borders (~50-100με). It is concluded that loading during the early stages of repair may impede stabilization of the injury site important for early bone matrix deposition, whereas loading while matrix deposition and remodeling are ongoing may enhance stabilization through the formation of additional cartilage and bone.